This proposal aims to determine the role of cytoplasmic dynein in the establishment and maintenance of microtubule polarity patterns in the axon. The nearly uniform plus-end distal orientation of microtubules is a hallmark characteristic of axons and is highly conserved in neurons found throughout nature. This microtubule polarity is critical for neuronal function, as motor proteins that carry cargoes such as organelles and vesicles use microtubule polarity to guide their movement. Yet since its discovery in 1981, little progress has been made in elucidating the underlying mechanism responsible for generating and preserving the axonal microtubule array. Cytoplasmic dynein, a minus-end directed motor protein, is capable of transporting microtubules with their plus- ends leading. The central hypothesis of this proposal is that dynein drives short microtubules into the axon with their plus-ends leading to build the microtubule array of the axon, but also drives minus-end distal microtubules out of the axon, to preserve fidelity of the axon's microtubule polarity pattern. Such minus-end distal microtubules may arise during plastic events such as axonal branch formation, or as a result of disease or injury-related challenges. While the majority of microtubule transport in the axon is anterograde, a notable fraction is retrograde, which is consistent with the existence of such a clearing mechanism. However, the role of cytoplasmic dynein has proven technically difficult to investigate because previous methods for inhibiting or depleting this moto protein did so gradually, thus introducing the potential for compensatory changes in the levels of other motor proteins. Additionally, traditional methods for visualizing microtubule transport were sub- optimal due to technical limitations. Here, the hypothesis will be tested through two specific aims - the first aim will determine the effects on axonal microtubule polarity orientation of inhibiting cytoplasmic dynein; the second aim will test the hypothesis that cytoplasmic dynein clears minus-end distal microtubules from the axon by transporting them back to the cell body. Proposed studies on these two aims utilize primary cultures of rat sympathetic neurons for cell biological analyses using innovative live-cell imaging techniques and sophisticated inhibition strategies that allow acute, reversible inhibition of dynein. These strategies circumvent longstanding technical issues, allowing the hypothesis to be tested, while simultaneously creating a new gold standard for observing microtubule transport in living neurons. The notion of dynein-driven transport of microtubules as being responsible for the establishment and maintenance of microtubule polarity patterns in the axon has profound implications for the understanding of neuronal development and the progression and potential treatments of neurological diseases and injuries.